Increasing glutathione levels for therapy

ABSTRACT

The present invention concerns compositions and methods related to utilizing glycine and N-acetylcysteine for a variety of methods, including, for example, reducing deleterious effects of oxidative stress; treating and/or preventing diabetes; and/or increasing GSH levels.

The present invention claims priority to U.S. Provisional PatentApplication Ser. No. 61/138,591, filed Dec. 18, 2008, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is directed to the fields of biochemistry, cellbiology, chemistry, molecular biology, and medicine.

BACKGROUND OF THE INVENTION

The free radical theory of aging suggests that the biological process ofaging results in increased oxidative stress in elderly humans. Theability of a cell to resist the damaging potential of oxidative stressis determined by a vital balance between generation of oxidant freeradicals and the defensive array of antioxidants available to the cell.There are multiple antioxidant defense systems and of these, glutathione(GSH) is the most abundant intracellular component of overallantioxidant defenses. GSH, a tripeptide, is synthesized from precursoramino-acids glutamate, cysteine, and glycine in two steps catalyzed byglutamate cysteine ligase (GCL, also known as γ-glutamylcysteinesynthetase, EC 6.3.2.2) and γ-L-glutamyl-L-cysteine:glycine ligase (alsoknown as gluta0thione synthetase, EC 6.3.2.3), and GSH synthesis occursde novo in cells.

Glutathione deficiency has been implicated in several diseases in humansincluding protein energy malnutrition in children, sickle-cell anemia,infection, neurological disorders such as Parkinson's disease, HIVinfections, liver disease and cystic fibrosis. Evidence from severalanimal (Stohs et al., 1984; Farooqui et al, 1987; Liu et al., 2000) andhuman studies (Al-Turk et al., 1987; Matsubara et al., 1991; Lang et a.,1992; Samiec et al., 1998; Erden-Inal et al., 2002; Loguercio et al.,1996) suggest that concentrations of glutathione also decline withaging. GSH deficiency in aging is associated with an increasedpro-oxidizing shift (Rebrin, 2008) leading to increased oxidative stress(Rikans and Hornbrook, 1997). These changes have been implicated indiseases of aging such as cataracts (Campisi et al., 1999; Castorina etal., 1992; Sweeney et al., 1998), age-related macular degeneration(Samiec, 1998), altered immune function (Fidelus and Tsan, 1987;Furukawa et al., 1987) and neurodegenerative disease (Liu et al., 2004),and in increased DNA damage (Hashimoto et al., 2008) at a molecularlevel. While the underlying mechanisms for aging-associated glutathionedeficiency is not well understood, there are suggestions thatperturbations in glutathione synthesis could be involved (Toroser andSohal, 2007).

Two key mechanisms for the intracellular GSH deficiency are suppressedsynthesis and/or increased consumption relative to synthetic capacity.To determine whether increased GSH consumption or suppressed synthesiswas responsible for intracellular GSH deficiency in aging, the inventorused an established stable-isotope tracer method (Reid and Jahoor, 2000)to measure in vivo erythrocyte GSH synthesis in 8 young and 8 agedhumans, before and after 14 days of supplementation of 2 key amino-acidprecursors of glutathione synthesis, cysteine and glycine. Glutathioneconcentrations within erythrocytes, and oxidative stress and markers ofoxidant damage within plasma, were also measured.

Other and further objects, features, and advantages will be apparentfrom the following description of the presently preferred embodiments ofthe invention, which are given for the purpose of disclosure.

SUMMARY OF THE INVENTION

In certain embodiments of the invention, the present invention concernscompositions and methods related to utilizing glycine andn-acetylcysteine (NAC) for therapeutic and/or preventative indicationsin mammals in need thereof. The mammals can be of any kind and caninclude humans, dogs, cats, horses, pigs, sheep, and goats, for example.In certain embodiments, the present invention is directed to one or moremethods and/or compositions that concern impaired glutathione turnoverand/or increased oxidative stress and/or oxidant damage in a mammal,including such impaired glutathione turnover and/or increased oxidativestress and/or oxidant damage in aging or diabetes. In specificembodiments, the present invention concerns beneficial effects ofdietary supplements with glycine and n-acetylcysteine in a mammal inneed thereof, including one that is aging or has diabetes, for example.

A mammal in need thereof can include one that needs prevention ortreatment of deleterious effects of aging or that needs prevention ortreatment of diabetes or complications from diabetes or that needsprevention or treatment from one or more of the following: dyslipidemia;insulin resistance; obesity; fatty acid oxidation; diabeticdyslipidemia; diabetic microvascular complications (for example,nephropathy, retinopathy, and/or neuropathy); high cholesterol and/ortriglyceride levels; fatty liver disease; neurodegenerative disease inaging; statin-induced myopathy.

In some embodiments, the present invention concerns individuals, forexample elderly humans, that have decreased GSH levels for any reason,including because of diminished synthesis, and in certain embodiments itis diminished because of poor availability of precursor amino acids, forexample. The low GSH state predisposes an individual to increasedoxidative stress, measured by plasma markers of oxidative damage, forexample. Dietary supplementation with both NAC and glycine results inimproved GSH synthesis and concentrations, and decreases in plasmamarkers of damage, in certain embodiments of the invention.

In one embodiment of the invention, there are methods and compositionsthat are useful for reducing and/or preventing oxidative stress in anindividual. In a specific embodiment, the methods and compositions areuseful for treating and/or preventing medical conditions associated withoxidative stress. In a particular embodiment, the methods andcompositions of the invention are useful for treating and/or preventingmedical conditions associated with reduced levels of glutathione. In onespecific embodiment of the invention, the methods and compositions areuseful for treating diabetes. In a certain aspect of the invention, themethods and compositions are useful for providing to the elderly. Inparticular cases, the present invention provides methods andcompositions useful for aging.

In certain embodiments, the invention concerns compositions and thefollowing exemplary method(s): method to reduce plasma F2-isoprostanelevels; method to reduce plasma F3-isoprostane and/or F2-isoprostanelevels (for example, as it relates to a marker for brain oxidativestress); method to increase GSH production; method to increase GSHintracellular concentration; method to increase liver (and separately,muscle, for example) GSH levels; method to improve insulin sensitivity;method to increase fat oxidation; method to reduce body weight; methodto treat/prevent dyslipidemia; method to treat/prevent fatty liverdisease; method to lower cholesterol level; method of preventingmyopathy, including statin induced myopathy; and/or method to lowertriglyceride level.

In particular embodiments, the present invention concerns improving atleast one symptom of, treating, and/or preventing the following: 1.dyslipidemia and insulin resistance; 2. obesity; 3. fatty acidoxidation; 4. diabetic dyslipidemia; 5. diabetic microvascularcomplications—nephropathy, retinopathy, neuropathy; 6. loweringcholesterol and triglyceride levels; 7. fatty liver disease; 8.neurodegenerative disease in aging; and/or 9. preventing statin-inducedmyopathy.

In one embodiment of the invention, there is a composition consistingessentially of glycine and N-acetylcysteine. In another embodiment ofthe invention, there is a composition consisting of glycine andn-acetylcysteine.

In certain embodiments of the invention, there is a method of reducingdeleterious effects of oxidative stress in an individual, comprising thesteps of providing an effective amount of glycine and N-acetylcysteineto the individual. In a specific embodiment, the individual is receivingtreatment or has received treatment for diabetes.

In an additional embodiment, there is a method of treating and/orpreventing diabetes in an individual, comprising the step of providingan effective amount of glycine and n-acetylcysteine to the individual.In specific cases of any aspect of the invention, the glycine andn-acetylcysteine are provided to the individual in the same compositionor different compositions. In some specific cases, the glycine andn-acetylcysteine are provided orally to the individual. In particularcases the glycine and n-acetylcysteine are provided to the individual inspecific ratio and/or by specific delivery regimen.

In one embodiment of the invention, there is a method of treating and/orpreventing diabetes in an individual, comprising the steps of: a)identifying an individual in need of diabetic treatment; and b)providing an effective amount of glycine and N-acetylcysteine to theindividual. In a particular embodiment of the invention, there is amethod of treating and/or preventing complications from diabetes in anindividual, comprising the steps of: a) identifying an individual inneed of preventing complications from diabetes; and b) providing aneffective amount of glycine and N-acetylcysteine to the individual.

In specific cases of the invention, there is a method of reducing plasmaF2-isoprostane levels, F3-isoprostane levels, or both in an individual,comprising the step of providing an effective amount of glycine andn-acetylcysteine to the individual.

In certain aspects, there is a method of increasing GSH production in anindividual, comprising the step of providing an effective amount ofglycine and n-acetylcysteine to the individual.

In particular aspects, there is a method of increasing GSH intracellularconcentration in an individual; increasing liver and/or muscle GSHlevels in an individual; improving insulin sensitivity in an individual;increasing fat oxidation in an individual; reducing body weight in anindividual; treating and/or preventing dyslipidemia in an individual;treating and/or preventing fatty liver disease in an individual;lowering cholesterol level in an individual; preventing myopathy in anindividual and/or lowering triglyceride level in an individual,comprising the step of providing an effective amount of glycine andn-acetylcysteine to the individual.

In certain embodiments of the invention, because aging is associatedwith impaired fat oxidation and obesity, and also with glutathionedeficiency due to impaired synthesis, providing glycine andn-acetylcysteine restores glutathione synthesis and concentrations, andalso improves fat oxidation, insulin resistance, obesity, and/ordyslipidemia.

In certain embodiments of the invention, an effective amount of glycineand n-acetylcysteine is provided to an individual for the improvedbiogenesis and/or mitochondrial function. In specific cases, aneffective amount of glycine and n-acetylcysteine is provided to anindividual for improving physical performance in the individual, such asimproved athletic performance. In certain aspects, the improvedmitochondrial biogenesis in the individual directly or indirectlyresults in improved athletic performance of the individual.

In certain embodiments of the invention, longevity is increased in anindividual that is provided an effective amount of glycine andn-acetylcysteine. Thus, in particular embodiments of the invention thereare methods and compositions related to increasing lifespan of anindividual. The individual may or may not have life-threatening medicalconditions. The individual has diabetes or obesity or other medicalconditions described herein, in certain embodiments.

In certain cases of the invention, providing an effective amount ofglycine and n-acetylcysteine improves PGC1a and/or AMPK levels. Inspecific cases, such an improvement provides therapeutic benefit inapplications related to insufficient levels of PGC1a and/or AMPK.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIGS. 1A-1B show erythrocyte GSH concentrations.

FIGS. 2A-2B show GSH fractional synthesis rate.

FIGS. 3A-3B show GSH absolute synthesis rate.

FIGS. 4A-4B show erythrocyte glycine flux.

FIGS. 5A-5B show plasma cysteine flux.

FIG. 6A demonstrates baseline data in diabetec vs. control subjects, andFIG. 6B shows GSH turnover in diabetics after precursor supplementation.

FIG. 7 illustrates a pathway for glutathione synthesis.

FIG. 8A shows basal GSH levels and precursor intake in young and elderlyhumans, and FIG. 8B shows GSH turnover in the elderly after precursorsupplementation.

FIG. 9 illustrates an exemplary study design.

FIG. 10 illustrates an exemplary infusion design.

FIG. 11 demonstrates human studies related to the role of GSH on fatoxidation, weight regulation, and insulin resistance.

FIG. 12 demonstrates animal studies related to the role of GSH on fatoxidation, weight regulation, and insulin resistance.

FIG. 13 shows that mice were matched for GTT levels between the oldcontrol PRE and Old RX GP-PRE in an exemplary study for aging associatedwith GSH deficiency.

FIG. 14 demonstrates body weight changes in an exemplary study for agingassociated with GSH deficiency.

FIG. 15 shows EE per mouse and per KG LBM in an exemplary study foraging associated with GSH deficiency.

FIG. 16 demonstrates fat oxidation in an exemplary study for agingassociated with GSH deficiency.

FIG. 17 shows ¹³C₁ palmitate oxidation in an exemplary study for agingassociated with GSH deficiency, wherein treated old mice have asignificantly higher oxidation of fat than untreated old mice, and evenexceed that of young mice.

FIG. 18 demonstrates GTT in an exemplary study for aging associated withGSH deficiency.

FIG. 19 shows data from an exemplary study for aging associated with GSHdeficiency, in particular glucose tolerance after 6 weeks. Treated oldmice have a significantly improved glucose tolerance (insulinresistance) compared to untreated old mice, and are similar now to youngmice.

FIG. 20 demonstrates ITT in an exemplary study for aging associated withGSH deficiency; in particular, insulin sensitivity after 6 weeks isdemonstrated. This graph shows that treated old mice have asignificantly improved insulin sensitivity compared to untreated oldmice and are similar now to young mice.

FIG. 21 demonstrates % ¹³C6-glucose oxidized in an exemplary study foraging associated with GSH deficiency.

FIG. 22 shows body composition in an exemplary study for agingassociated with GSH deficiency.

FIG. 23 shows that when precursors are provided in the diets, synthesisof glutathione increases significantly to that seen in non-diabetichumans, together with a significant fall in oxidative stress and markersof oxidative damage.

FIG. 24 shows that improving GSH also results in increasing fatoxidation and decreasing fatty acid concentrations in humans withuncontrolled diabetes.

FIG. 25 demonstrates that improving GSH also results in decreasing F3isoprostanes (FIG. 25A) and decreasing F2 isoprostanes (FIG. 25B) inelderly humans.

FIG. 26 shows that improving glutathione lowers total cholesterol andtriglyceride levels in mice.

FIG. 27 shows basal body weights in certain groups of mice.

FIG. 28 shows basal whole-body fat oxidation in young mice, control oldmice, and experimental old mice that have not received treatment.

FIG. 29 illustrates fat oxidation by calorimetry of young mice versusuntreated old mice and treated old mice.

FIG. 30 illustrates the AMPK signaling cascade.

FIG. 31 illustrates phosphorylated AMPK to AMPK ratio in the liver.

FIG. 32 shows the ratio of phosphorylated ACC to ACC in the liver.

FIG. 33 shows energy metabolism in white muscle via PGC1-a levels in thetested mice.

FIG. 34 demonstrates insulin response to GTT in the treated micecompared to controls. GTT is ‘Glucose tolerance test’ and the product ofthe fasting plasma glucose (FPG) and fasting insulin (FI) provides anindex of insulin sensitivity.

FIG. 35 shows ¹³C₆-glucose oxidation in young versus old mice.

FIG. 36 shows ¹³C₆-glucose oxidation in treated old mice compared toyoung and untreated old mice.

FIG. 37 shows body composition in treated versus control mice.

FIG. 38 shows GSH concentrations in liver and muscle of young, controlold, and treated mice.

FIG. 39 shows respiratory quotient (RQ) in treated elderly humanscompared to elderly controls and young controls.

FIG. 40 demonstrates fat oxidation in young humans compared to elderlyhumans either treated with dietary supplementation of the invention ornot treated.

FIG. 41 shows fasting plasma fatty-acid concentrations in young humans,elderly untreated humans (EH-B) and elderly treated humans (EH-GC).

FIG. 42 shows fasting plasma glucose in young humans compared to treatedand untreated elderly humans.

FIG. 43 shows plasma concentrations of creatine kinase in treated anduntreated old mice, and young mice.

FIG. 44 demonstrates RBC glutathione concentrations in treated anduntreated uncontrolled diabetics.

FIG. 45 shows GSH fractional synthesis rate in treated and untreateduncontrolled diabetics.

FIG. 46 demonstrates oxidative stress and oxidant damage in treateddiabetic and nondiabetic humans.

FIG. 47 shows improving GSH also results in increasing fat oxidation anddecreasing fatty acid concentrations in humans with uncontrolleddiabetes.

FIG. 48 shows energy metabolism in diabetic mice.

DETAILED DESCRIPTION OF THE INVENTION VI. Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

As used herein, the term “complications from diabetes” in specificembodiments refers to diabetic nephropathy, neuropathy, retinopathy,diabetic obesity, diabetic dyslipidemia, cardiometabolic syndrome, andcombinations thereof, for example.

As used herein, the term “effective amount” refers to an amount ofglycine and n-acetylcysteine that is required to improve at least onesymptom of a medical condition in an individual; in specificembodiments, the medical condition exists in the individual directly orindirectly because of insufficient levels of glutathione. In specificembodiments, the effective amount refers to the amount of glycine andn-acetylcysteine that is utilized to increase glutathione levels in theindividual.

As used herein, the term “elderly” refers to an individual over the ageof at least 60 years of age.

As used herein, the term “oxidative stress” refers to the state in anindividual, or cell or tissue of an individual, of an imbalance betweenthe production of reactive oxygen and the ability to detoxify thereactive intermediates or easily repair the resulting damage in abiological system. The natural reducing environment within cells ismaintained by processes using a constant input of metabolic energy, anddisturbances in this normal redox state can result in toxic effectsthrough the production of, for example, free radicals and peroxides thatdamage cellular components, such as proteins, lipids, and/or DNA, forexample.

VII. Certain Embodiments of the Present Invention

In certain embodiments of the invention, there are methods andcompositions for the treatment of medical conditions caused directly orindirectly by insufficient GSH levels in the individual. The individualmay be of any age or state of health, although in particular embodimentsthe individual is elderly, is susceptible to particular medicalconditions associated directly or indirectly with insufficient GSHlevels, or has a medical condition that is associated directly orindirectly with insufficient GSH levels. The compositions delivered tothe individual in such cases include at least glycine andn-acetylcysteine, in particular as precursor amino acids to facilitateraising glutathione levels in the individual

In some aspects of the invention, an individual is treated fordeleterious effects of aging. Aging and certain medical conditions areassociated with increased oxidative stress, but the underlyingmechanisms are unknown. In certain aspects of the invention, in vivokinetics of GSH were measured to evaluate whether changes in glutathioneturnover could account for the elevated oxidative stress in aging, andwhether stimulating glutathione synthesis using dietary amino acidprecursors could reduce aging-associated oxidative stress. In exemplarycases, the inventor used a primed infusion of [²H₂]glycine to measureintracellular GSH synthesis in vivo in 8 non-diabetic elderly humans and8 healthy controls. The elderly subjects were studied a second timeafter 2-weeks of dietary supplementation with n-acetylcysteine(as acysteine donor) and glycine, as GSH precursors. Plasma oxidative stress,markers of oxidant damage, and dietary intakes of protein were alsomeasured. Compared to young controls, elderly subjects had significantlylower erythrocyte GSH concentrations (83.14±6.43 vs. 43.93±6.21%/d,p<0.001) fractional synthetic rates (2.08±0.12 vs. 1.12±0.18 mmol/L.RBC,p<0.001) and absolute synthetic rates (1.73±0.16 vs. 0.53±0.12mmol/L.RBC/d, p<0.0001) in the basal state. This was associated withsignificantly increased plasma markers of oxidative damage and reactiveoxygen metabolites (309± vs. 332±303 in the elderly subjects compared tocontrols. After supplementation there were marked and significantincreases in erythrocyte GSH concentrations, fraction and absolutesynthetic rates to that of young controls, together with a fall inreactive oxygen metabolites and plasma F2-isoprostanes again to that ofyoung humans. These data indicate that elderly humans are GSH deficient,and that this occurs because of suppressed synthesis. Providingprecursor supplements in the diet leads to not only to restoration ofGSH concentrations primarily due to an increase in the fractionalsynthetic rate to that of young humans, but also in a striking reductionin oxidative stress and in oxidant damage to that of young healthycontrols.

VIII. Pharmaceutical Compositions

In particular embodiments, the present invention is directed topharmaceutical compositions for use in treating and/or preventingdeleterious effects of aging, diabetes, complications from diabetes,obesity, dyslipidemia, high cholesterol levels, high triglyceridelevels, and so forth. In specific embodiments, glycine is administeredat 1.33 mmol/kg/d and NAC is administered at 0.83 mmol/kg/d for aparticular period of time. Durations of treatment may last for 1 week, 2weeks, 3 weeks, one month, two months, three months, four months, fivemonths, six months, one year, two years, five years, ten years, fifteenyears, twenty years, twenty-five years, thirty years, and so forth, forexample. In some cases the treatment lasts for the remaining life of theindividual. In specific embodiments, the administration occurs until nodetectable symptoms of the medical condition remain. In specificembodiments, the administration occurs until a detectable improvement ofat least one symptom occurs and, in further cases, continues to remainameliorated.

Where the invention is directed to treating with the compounds of thepresent invention, administration of the compounds of the invention witha suitable pharmaceutical excipient as necessary can be carried out viaany of the accepted modes of administration. The compounds may becomprised in a pharmaceutically acceptable excipient, which may beconsidered as a molecular entity and/or composition that does notproduce an adverse, allergic and/or other untoward reaction whenadministered to an animal, as appropriate. It includes any and/or allsolvents, dispersion media, coatings, antibacterial and/or antifungalagents, isotonic and/or absorption delaying agents and/or the like. Theuse of such media and/or agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media and/oragent is incompatible with the active ingredient, its use in thetherapeutic compositions is contemplated.

Thus, administration can be, for example, intravenous, topical,subcutaneous, transcutaneous, intramuscular, oral, intra-joint,parenteral, peritoneal, intranasal, intravesical or by inhalation.Suitable sites of administration thus include, but are not limited to,skin, bronchial, gastrointestinal, anal, vaginal, eye, bladder, and ear.The formulations may take the form of solid, semi-solid, lyophilizedpowder, or liquid dosage forms, such as, for example, tablets, pills,capsules, powders, solutions, suspensions, emulsions, suppositories,retention enemas, creams, ointments, lotions, aerosols or the like,preferably in unit dosage forms suitable for simple administration ofprecise dosages.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, and the like. Preferably, the composition will beabout 5% to 75% by weight of a compound or compounds of the invention,with the remainder consisting of suitable pharmaceutical excipients.Appropriate excipients can be tailored to the particular composition androute of administration by methods well known in the art, e.g.,REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co.,Easton, Pa. (1990).

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. The composition may take the form of a solution, suspension,tablet, pill, capsule, powder, sustained-release formulation, and thelike.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet or capsule, and thus, the composition can contain, alongwith the biologically active conjugate, any of the following: a diluentsuch as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.

The active compounds of the formulas may be formulated into asuppository comprising, for example, about 0.5% to about 50% of acompound of the invention, disposed in a polyethylene glycol (PEG)carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).

Liquid compositions can be prepared by dissolving or dispersing compound(about 0.5% to about 20%), and optional pharmaceutical adjuvants in acarrier, such as, for example, aqueous saline (e.g., 0.9% w/v sodiumchloride), aqueous dextrose, glycerol, ethanol and the like, to form asolution or suspension, e.g., for intravenous administration. The activecompounds may also be formulated into a retention enema.

If desired, the composition to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, such as, for example, sodium acetate,sorbitan monolaurate, or triethanolamine oleate.

For topical administration, the composition is administered in anysuitable format, such as a lotion or a transdermal patch. For deliveryby inhalation, the composition can be delivered as a dry powder (e.g.,Inhale Therapeutics) or in liquid form via a nebulizer.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences, supra., and similar publications. The composition to beadministered will, in any event, contain a quantity of the pro-drugand/or active compound(s) in a pharmaceutically effective amount forrelief of the condition being treated when administered in accordancewith the teachings of this invention.

Generally, the compounds of the invention are administered in atherapeutically effective amount, i.e., a dosage sufficient to effecttreatment, which will vary depending on the individual and conditionbeing treated. Typically, a therapeutically effective daily dose is from0.1 to 100 mg/kg of body weight per day of drug. Most conditions respondto administration of a total dosage of between about 1 and about 30mg/kg of body weight per day, or between about 70 mg and 2100 mg per dayfor a 70 kg person.

Stability of the conjugate can be further controlled by chemicalalterations, including D amino acid residues in the polypeptide chain aswell as other peptidomimetic moieties. Furthermore, stability of theconjugates could also be enhanced by unnatural carbohydrate residues.

The glycine and N-acetylcysteine components may be formulated in aparticular ratio. In certain embodiments, the formulation may comprisethe components in the following exemplary ratios: 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40,1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100,1:150, 1:200, 1:300, 1:400, 1:500, 1:600, 1:750, 1:1000, 1:10,000, andso forth, for example. In particular embodiments, the formulation maycomprise the components in the following percentages by formulation(either the same or different percentages for each): 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%, for example.

IX. Combination Treatments

Alternatively, the treatment of the invention may precede, follow, orboth another treatment by intervals ranging from minutes to weeks. Inembodiments where the inventive composition and the other agent areapplied separately to a cell of the individual, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the inventive composition and the otheragent would still be able to exert an advantageously combined effect onthe cell. In such instances, it is contemplated that one may contact thecell with both modalities within about 12-24 h of each other and, morepreferably, within about 6-12 h of each other. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, for example, wherein the inventivetreatment is “A” and the secondary agent for the medical condition ofthe invention as described herein, such as diabetic treatment (forexample only), is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the inventive compositions of the present invention toa patient will follow general protocols for the administration of drugs,taking into account the toxicity, if any, of the molecule. It isexpected that the treatment cycles would be repeated as necessary. Italso is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the describedtherapy.

X. Kits

Therapeutic kits associated with the compositions of the presentinvention comprise another aspect of the present invention. Such kitswill generally contain, in suitable container means, an inventivecomposition of the present invention. The kit may have a singlecontainer means that contains the inventive composition or it may havedistinct container means for the inventive composition and otherreagents that may be included within such kits.

The components of the kit may be provided as liquid solution(s), or asdried powder(s). When the components are provided in a liquid solution,the liquid solution is an aqueous or non-aqueous solution, with asterile aqueous or non-aqueous solution being particularly preferred.When reagents or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

The container means will generally include at least one vial, test tube,flask, bottle, syringe or other container means, into which thecomposition may be placed, and preferably suitably aliquoted. Where asecond agent is provided, the kit will also generally contain a secondvial or other container into which this agent may be placed. The kits ofthe present invention will also typically include a means for containingthe agent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained, for example.

In the kit of the invention, the glycine and the N-acetylcysteine may beprovided separately or in a mixture together.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow presenttechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exemplary Materials and Methods

The present example concerns exemplary materials and methods for methodsand/or compositions of certain embodiments of the present invention.

Subjects: The study was approved by the Institutional Review Board forHuman Studies at Baylor College of Medicine, and the procedures followedwere in accordance with the ethical standards of the institution. Tenhealthy elderly humans (60-75 years) were recruited into the aged arm,and ten younger subjects (20-40 years) were recruited as young controls.The elderly subjects were free of diabetes mellitus, thyroid disorders,hypercortisolemia, liver or renal impairment, and had no opportunisticinfections or illnesses for 6 months. All had sedentary lifestyles andnone consumed unusual diets or dietary supplements.

Subjects had a 4 day habitual diet assessment by a qualified dietitianwhere they charted and weighed all food consumed over a 4 day periodprior to the study.

Subjects had an initial visit to measure blood counts, glucoseconcentrations, liver and renal profiles.

Metabolic study protocol: Subjects were studied in the adult GeneralClinical Research Center (GCRC) of Baylor College of Medicine afterobtaining written informed consent. The protocol consisted ofintravenous infusions of stable isotopes to measure glutathionesynthesis in the fasted state, before and after 14 days of dietarysupplementation with glutathione precursor amino acids cysteine (asn-acetylcysteine) and glycine. Subjects were asked to consume theirusual habitual diets, and avoid any alcohol from one week beforebeginning the study to the end of the study period. They were fasted for10 h before the start of the stable isotope infusions.

The primary outcome variables were fractional and absolute syntheticrates of glutathione, plasma F2-isoprostane and lipid peroxide levels,and plasma oxidative stress measured as diacron reactive oxygenmetabolites (DROMS).

Stable Isotope protocol: A sterile solution of [²H₂]glycine (CambridgeIsotope Laboratories, Woburn, Mass.) was prepared in saline. On themorning of the infusion day, intravenous catheters were inserted intosuperficial veins of both arms, one for continuous infusion of thetracer solution and the other for repeated blood sampling. After a 20-mlblood sample prior to infusions was drawn, a bolus intravenous infusionof [²H₂]glycine (20 μmol/kg) was given to prime the glycine poolfollowed by a constant infusion of the same isotope at the rate of 15μmol·kg⁻¹·h⁻¹, and maintained for 8 h. Additional 5-ml blood sampleswere taken at 5, 6, 7, and 8 h for measurement of erythrocyteGSH-derived glycine isotopic enrichments.

Sample Analyses:

Blood chemistries. Baseline blood samples were collected. The samplesfor plasma analyses were immediately centrifuged, and plasma separatedand frozen in a −80 C freezer for later analyses. Hemoglobin, hemotocritand red blood cell counts were measured. Oxidative stress was measuredusing a diacron Reactive Oxygen Metabolites (DROMS) kit. Plasmachemistries, lipid peroxides and F2-isoprostanes were measured asoutlined below. An additional tube was collected for measurement of F2isoprostanes.

Erythrocyte GSH analyses. Erythrocyte GSH concentration and isotopicenrichment were measured in duplicate 1-ml aliquots of whole bloodcollected. Briefly, one sample was mixed immediately in a cryotube with0.5 ml of chilled, isotonic monobromobimane (MBB) buffer solution (pH7.5) containing the following (in mmol/l): 5 MBB, 17.5 Na₂EDTA, 50potassium phosphate, 50 serine, and 50 boric acid. The whole blood-MBBmixture was centrifuged at 1,000 g for 10 min at 4° C., and then thesupernatant fluid was incubated in the dark for 20 min for developmentof the plasma GSH-MBB derivative. Another 1.0 ml of MBB buffer was addedto the packed erythrocytes, which were immediately lysed by rapid freezeand thaw with liquid nitrogen, and the lysed erythrocyte-MBB buffermixture was shaken and left in the dark at room temperature for 20 minfor development of the erythrocyte GSH-MBB derivative. Proteins wereprecipitated by using 0.5 ml of 2 mol/l perchloric acid, and thesupernatant fluids were stored at −70° C. until further analysis.

Erythrocyte GSH was isolated and the concentration measured using aWaters HPLC system using a 717plus autosampler complexed to a Waters2475 fluorescent detector and equipped with a reverse-phase ODS Hypersilcolumn (5 μm, 4.6×200 mm; Waters Inc.,). Elution of the GSH wasaccomplished with a 3-13.5% acetonitrile linear gradient in 1% aceticacid (pH 4.25) at a flow rate of 1.1 ml/min. The GSH elution wascollected using a fraction collector, dried, and hydrolyzed for 4 h in 4mol/l HCl at 110° C. (REFS).

Erythrocyte free glycine was extracted from the protein-free supernatantby cation exchange chromatography. Erythrocyte free glycine anderythrocyte GSH-derived glycine were converted to the n-propyl esterheptafluorobutyramide derivatives, and the isotope ratio of each wasmeasured by negative chemical ionization gas chromatography massspectrometry on an Agilent 6980 gas chromatograph complexed to a 5973mass spectrometer (Agilent Technologies, Wilmington, Del.), monitoringions at mass to charge ratio (m/z) 293 to 295.

Oxidant stress. The D-ROMS test was used to measure plasma hydroperoxideconcentration as an index of free radical formation. This test is basedon the concept that the amount of organic hydroperoxides present inplasma is related to the free radicals from which they are formed.Briefly, plasma is reacted with an acidic acetate buffer (pH 4.8), whichliberates transition metal ions that catalyze the decomposition of thehydroperoxides to alkoxy and peroxyl radicals. These newly formedradicals in turn oxidize the spectrophotometric marker(N,N-diethyl-p-phenylenediamine), which is detectable at an absorptionat 505 nm as U.CARR. (carratelli units). One U.CARR. is equal to 0.8mg/l, hydrogen peroxide.

Plasma markers of Oxidant damage (F2-isoprostanes and lipid peroxides):F2-Isoprostanes. Blood (4 mL) was collected into a lithium-heparin tubecontaining indomethacin (15 μmol/L). After centrifugation at 2400 g for10 min at 4° C. to separate plasma and erythrocytes, aliquots of plasma(1 mL) were transferred to Eppendorf tubes containing butylatedhydroxytoluene, at a final concentration of 20 μmol/L. One milliliter of1 mol/L KOH was added to hydrolyze the plasma sample at 40° C. for 30min to release bound lipids. Then, 1 mL of 1 mol/L HCl and 2 mL of 100mmol/L formate buffer (pH 3.0) were added and the sample centrifuged at2400g for 10 min; the supernatant then underwent solid-phase extractionwith the addition of iPF2{alpha}-III-d4 as an internal standard. AnOasis HLB extraction cartridge preconditioned with methanol and 10mmol/L formate buffer (pH 3.0) was used for isoprostane extraction byfirst washing with 5 mL of the formate buffer followed by 5 mL ofacetonitrile-water (15:85 by volume), and then F2-isoprostanes wereeluted by washing the cartridge with 2 mL of hexane-ethylacetate-propan-2-ol (30:65:5 by volume). F2-iPs were then analyzed bygas chromatography- mass spectrometry (GC/MS). After evaporation undernitrogen, the extracted F2-iPs in the samples were incubated with 25 μLof pentafluorobenzyl bromide (100 mL/L in acetone) and 25 μL ofN,N-diisopropylethylamine (200 mL/L in acetone) at 60° C. for 10 min.The resulting pentafluorobenzyl ester was incubated with 50 μL ofN,O-bis-(trimethylsilyl)trifluoroacetamide and 5 μL ofN,N-diisopropylethylamine (200 mL/L in acetone) at 60° C. for 5 min, anddried under nitrogen. The pentafluorobenzyl-trimethylsilyl derivativeswere reconstituted in 40 μL of isooctane and the samples analyzed on anAgilent 6890 series gas chromatograph (in NCI mode) coupled to a 5973mass spectrometer (Agilent Technologies, Wilmington, Del.) using methaneas the reagent gas. Chromatography was carried out on an SPB-1701 column(30 m×0.25 mm; film thickness, 0.25 μm; Supelco Inc.) using helium asthe carrier gas. Selected ion monitoring was performed to monitor them/z 569 and 573 for F2-iPs and the internal standard, respectively. Peakidentification was based on the comparison of the relative retentionindices with the internal standard, and the concentration of F2-iPisomers in the samples was calculated using the ratio of the peak heightof m/z 569 to that of m/z 573.

Lipid peroxides. Were measured using a standard kit.

Dietary Assessment

Subjects were issued with a food weighing scale and weighed andmaintained a 4-day food diary during the week prior to the infusionexperiment with verbal and written instructions to add to their diaryevery time they ate or drank, describing the food/drink as accurately aspossible and weighing accurately the amounts of food consumed. The diaryconsisted of three weekdays and one weekend day. All of the diaries werecompleted and collected when the subjects were admitted for the infusionexperiments. The diaries were analyzed using Nutritionist Five (SanBruno, Calif.) software, by a trained nutritionist.

Calculations

The fractional synthesis rate of erythrocyte GSH (FSRGSH) per day wascalculated according to the precursor-product equation as describedbelow

FSRGSH (%/day)=(IRt7−IRt5)/(IRrbc×1200/t7−t5)

where IRt7−IRt5 is the increase in the isotope ratio of erythrocyteGSH-bound glycine between the fifth and seventh hours of infusion, whenthe isotope ratio of erythrocyte free glycine, IRrbc, had reached asteady state. The units of FSR are percent per day (%/day).

The absolute synthesis rate (ASR) of erythrocyte GSH per day wascalculated as.

ASR=Erythrocyte GSH concentration×FSR

Where FSR is the fractional synthesis rate. The units of ASR areexpressed as millimoles per liter per day of packed erythrocyte.

Statistics

Data are expressed as means±SD. Differences in means between the SCDgroup and the control group were determined using an independent t-testwith the Satterthwaite adjustment for unequal variances whereappropriate. For variables where the distributions were skewed, thetwo-sample Wilcoxon test was used. Differences in net tracer-to-traceeratio between groups at plateau were determined by repeated-measuresanalysis of variance. Data analysis was performed with the Statmatestatistical software, version 8.2, for Windows (Stata, College Station,Tex.). Results were considered to be statistically significant ifP<0.05.

Example 2 The Effects of Aging on Glutathione Synthesis and OxidativeStress Baseline Characteristics

By design, the ages of the two groups were different as the study groupswere aged subjects (60-75 years) and the control group were youngsubjects (20-40 years). Younger subjects were lighter and had lower bodymass indexes compared with aged subjects. There were no differencesbetween the two groups in hematocrit, hemoglobin concentrations, renalfunctions or liver enzymes. Both groups were non-diabetic, but agedsubjects were more insulin resistant with significantly higherconcentrations of fasting glucose and glycosylated hemoglobin (Table 1).

TABLE 1 Baseline clinical, hematological and biochemical characteristicsParameters Young controls Elderly P Age (years) 39.8 ± 1.0 70.3 ± 2.4<0.01 Weight 73.9 ± 2.1 82.9 ± 5.4 NS BMI 25.7 ± 0.6 29.8 ± 1.4 <0.05 Hb(g/L) 14.2 ± 0.3 13.7 ± 0.3 NS Hematocrit (%) 42.4 ± 0.7 41.0 ± 0.8 NSFasting plasma  4.9 ± 0.2  5.9 ± 0.3 <0.05 glucose (mmol/L) HbA1c (%) 5.2 ± 0.1  5.7 ± 0.1 <0.01 BUN (mg/dl) 13.8 ± 1.0 13.1 ± 1.0 NSCreatinine (mg/dl)  1.0 ± 0.0  0.9 ± 0.1 NS ALT (U/L) 22.6 ± 3.2 28.1 ±1.7 NS AST (U/L) 28.1 ± 4.4 25.8 ± 1.5 NS

In the aged group alone, there were no differences in hematologicparameters, renal functions of liver enzymes before and after GSHprecursor supplementation (Table 2).

TABLE 2 Serum biochemistry pre- and post- supplementation ParametersPre-treatment Post-treatment P Hb (g/L) 13.7 ± 0.3 13.8 ± 0.2 NSHematocrit (%) 41.0 ± 0.8 41.5 ± 0.6 NS BUN (mg/dl) 13.1 ± 1.0 13.5 ±1.1 NS Creatinine (mg/dl)  0.9 ± 0.1  0.9 ± 0.0 NS ALT (U/L) 28.1 ± 1.728.4 ± 1.4 NS AST (U/L) 25.8 ± 1.5 25.6 ± 1.2 NS

Table 3 demonstrates the oxidant concentrations in all subject.

TABLE 3 Oxidant concentrations in all subjects Young ParametersElderly-Pre Elderly-Post Controls P DROMs 346 ± 6^(∞)  277 ± 20*  304 ±16 *<0.05 (mmol/L) ^(∞)<0.05 F2 Isoprostane 136.3 ± 11.3* 84.8 ±11.1^(∞) 97.2 ± 8.3 *<0.05 (pg/ml) ^(∞)<0.05 Lipid Peroxide  5.90 ±1.01* 2.95 ± 0.63^(∞)  1.92 ± 0.28 *<0.01 (umol/L)  ^(∞)<0.001 *Youngcontrols vs. Aged: Pre-supplementation ^(∞)Aged: Pre-supplementation vs.Aged: Post-supplementation

Erythrocyte and Plasma GSH Kinetics

Compared with young controls, aged humans had 46.2% lower erythrocyteGSH concentrations (mean±SE, 1.12±0.18 vs. 2.08±0.12 mmol/L.RBC, P<0.01)(FIG. 1 a). Aged humans also had 44.9% lower GSH FSR (mean±SE,45.80±5.69 vs. 83.14±6.43, % day-1, P<0.001), and 68.2% lower ASR(mean±SE, 0.55±0.12 vs. 1.73±0.16, mmol.LRBC-1.day-1, P<0.01), (FIGS. 2a and 3 a). The erythrocyte glycine flux (mean±SE, 560.0±43.5 vs.769.1±47.9, umol.kg-1.h-1, P<0.05) and plasma cysteine flux (mean±SE,46.1±4.5 vs. 59.3±2.4, umol.kg-1.h-1, P<0.05) were also 27.2% and 22.32%lower in the aged humans compared to young controls (FIG. 4 a).

Supplementation with GSH precursor amino acids cysteine (asn-acetylcysteine) and glycine for 14 days led to 94.6% improvement inerythrocyte GSH concentrations (mean±SE, 1.12±0.18 vs. 2.18±0.35mmol/L.RBC, P<0.001) and 78.8% higher FSR (mean±SE, 45.80±5.69 vs.81.91±7.70, % day-1, P<0.001), resulting in 230.9% increase in ASR(mean±SE, 0.55±0.12 vs. 1.82±0.39, mmol.LRBC-1.day-1, P<0.0001). Theerythrocyte Glycine flux (mean±SE, 560.0±43.5 vs. 769.1±47.9,umol.kg-1.h-1, P<0.05) increased by 28.1%, and the plasma cysteine fluxmean±SE, 46.1±4.5 vs. 59.3±2.4, umol.kg-1.h-1, P<0.05) by 26.7%. Therewere no significant differences between the young controls and the postsupplementation values of any of these parameters, suggesting that inaged humans, these values were restored to those seen in young controls(FIGS. 1-4 b).

Oxidant Parameters

The lower rates of synthesis of GSH in aged subjects were associatedwith significantly higher concentrations of markers of oxidative damage(plasma D-ROMs, plasma F2-isoprostanes and lipid peroxides; Table 2),compared to young controls. After 14 days of dietary supplementations,there was a significant fall in these parameters in the aged humans,with no differences between the young group and the GSH-replete agedgroup post-supplementation.

Significance of Present Exemplary Embodiments

In this study the inventor examined (1) the effect of aging onglutathione synthesis and oxidative stress by comparing in vivosynthesis and concentrations of glutathione within erythrocytes in youngand aged humans, plasma oxidative stress and plasma markers of oxidantdamage; and (2) the ability of aged humans to correct the defect in GSHsynthesis, when their diets were supplemented with GSH precursorproteins for 14 days, and the effect of this improvement on oxidativestress and markers of oxidant damage in the plasma. These datademonstrate that erythrocyte glutathione synthesis and concentrationsare markedly reduced in the aged humans compared to younger controls.Glutathione deficiency in aging humans is also associated with anincreased oxidant stress and in plasma markers of oxidant damage. Whenthe aged subjects were re-studied after a 14 day period of oral dietarysupplementation with two key precursor amino-acids of glutathionesynthesis, cysteine (as n-acetylcysteine) and glycine, both thefractional and absolute synthetic rates of glutathione increased to thatseen in young controls. This resulted in restoration of erythrocyteglutathione concentrations to that seen in young controls, and also ledto significant falls in both oxidative stress and plasma markers ofoxidant damage, again to levels found in young controls. These findingsindicate that the primary reason for glutathione deficiency in aginghumans is diminished synthetic capacity, and predisposes to decreasedability to combat the unopposed increase in oxidative stress, resultingin oxidant damage. The study also demonstrates that the novel use ofsimple, inexpensive dietary protein precursors to boost glutathionesynthesis can both restore glutathione concentrations, and also loweroxidative stress and decrease oxidant damage, in effect restoring theoxidant-antioxidant balance to that observed in young humans.

Several diseases of aging are associated with increased oxidative stressand increased oxidant damage (Samiec et al., 1998), (Campisi et al.,1999; Castorina et al., 1992; Sweeney and Truscott, 1998; Fidelus andTsan, 1987; Furukawa et al., 1987; Liu et al,. 2004) but the underlyingmechanisms are not well understood. Although Glutathione is the largestcomponent of intracellular antioxidants, and glutathione deficiency hasbeen reported in aging (Rizvi and Maurya, 2007), to the knowledge of theinventor no prior studies have examined fractional and absolutesynthetic rates of glutathione to understand the mechanistic correlationwith its intracellular concentrations in aging. One plausibleexplanation for Glutathione deficiency with aging is abnormalities inprotein turnover. Glutathione is a tripeptide comprised of glutamine,cysteine and glycine. As amino acid catabolism and recycling traces itspathway through glutamine as an intermediary, the inventor also examinedcysteine and glycine turnover to evaluate decreased GSH synthesis.Compared to younger controls, the turnover rate of glycine withinerythrocytes was markedly diminished, and the turnover of cysteinewithin the plasma was also significantly decreased. This observation isespecially interesting as both these amino-acids are ‘non-essential’amino acids, and the body retains the ability to synthesize cysteine andglycine, which suggests that protein turnover could be decreased inaging. The process of aging is associated with altered protein turnover(Young, 1990; Boirie et al., 1997; Morais et al., 1997; Fereday et al.,1997; Campbell et al., 1997). In addition, in vivo kinetic studies haveshown that when normal adults are fed diets either deficient in sulfuramino-acids(Lyons et al., 2000), or containing reduced amounts ofprotein (Jackson et al., 2004) GSH turnover is suppressed. Studies inanimal models show that absolute dietary deficiencies of GSH precursoramino acids, especially cysteine, can result in decreased GSHconcentrations (Grimble et al., 1992; Jahoor et al., 2005; Bella et al.,1999; Cresenzi et al., 2003). Therefore, the available evidenceindicates a role for reduced substrate availability to adversely affectin vivo glutathione synthesis with aging, resulting in intracellularglutathione deficiency.

In vivo synthesis of cysteine depends on adequate availability of itsprecursors, methionine and serine. Methionine is an essential(indispensable) amino acid that is supplied by the diet. Cysteinesynthesis occurs primarily in the liver from methionine and serine viathe transmethylation-transsulfuration pathway, where methionine isconverted to homocysteine, which in turn combines with serine to formcystathionine. The latter is then converted into cysteine and2-ketobutyrate by cystathionine L-homocysteine lyase. Thus, decreasedavailability of methionine and serine, impairment of thetransmethylation-transsulfuration pathway, and/or deficient dietaryintake of cysteine or gastrointestinal malabsorption may lead tocysteine deficiency. In addition, it has also been argued that anon-specific increase of protein intake in aging could lead to unwantedconsequences such as hyperhomocystinemia. As homocysteine production isupstream of cysteine, providing n-acetylcysteine directly supplementscysteine without a parallel increase in homocysteine. This study foundmarkedly decreased turnover of both cysteine and glycine in elderlyhumans, which improved significantly with just 14 days of precursorsupplementation.

This constellation of findings indicate that deficiency of GSHprecursors predisposes to GSH deficiency, elevated oxidative stress andoxidant damage in aging. To answer the key question of whethersupplementing the diets of elderly humans with GSH precursors cysteineand glycine would reverse the age dependent glutathione deficiency, theinventor addressed the same cohort of elderly humans after 14 days oforal dietary supplements. This resulted in significantly increasedturnover rates of glycine and cysteine within the erythrocyte and plasmarespectively. In turn, there was a significant increase in thefractional synthetic rate and the absolute synthetic rates of GSH,leading to restoration of erythrocyte GSH concentrations to that seen inyoung controls. The improved GSH synthesis and concentrations led to adramatic reduction in oxidative stress, and markers of oxidant damagedown to the levels observed in the younger controls.

Thus, in particular embodiments of the invention, severe glutathionedeficiency underlies the increased oxidative stress and oxidant damageassociated with human aging. Glutathione deficiency in aged humansoccurs primarily due to a markedly diminished capacity to synthesizeglutathione, as a result of limited availability of precursor aminoacids cysteine and glycine. Supplementing the diets of at least elderlyhumans, for example, with these amino-acids for 14 days boostsglutathione synthesis to that seen in young humans and restoresintracellular glutathione concentrations, resulting in significantreductions in oxidative stress and oxidant damage.

Example 3 The Effect of Diabetes on Glutathione Synthesis and OxidativeStress

Increased oxidative stress in poorly controlled diabetes is stronglylinked to diabetic complications. Levels of antioxidant Glutathione(GSH) are lower in diabetics, indicative of impaired antioxidantdefenses, and associated with increased levels of damaging lipidperoxides (ROOH). The inventor characterized whether (1) GSHconcentrations are lower due to decreased GSH synthesis; and (2) dietarysupplementation with GSH precursors (cysteine (as NAC) and glycine)would improve GSH synthesis, and decrease plasma markers of oxidativedamage (ROOH).

Eight diabetics were studied before and after 2-week dietarysupplementation with either n-acetylcysteine(NAC), glycine, or both.Subjects received infusions of ²H₂-glycine to measure the concentration,fractional synthetic rates (FSR) and absolute synthetic rates (ASR) ofred cell GSH. Plasma ROOH and glucose levels were measured. Eightnon-diabetic subjects were studied as controls. FIG. 9 illustrates anexemplary study design, and FIG. 10 shows an exemplary infusion design.

Results (Mean±SE): Diabetic subjects had a significantly lower GSHconcentration (1.17±0.05 v 2.01±0.05 mmol/LRBC, p<0.001), FSR (36±5 v75±2%/d, p<0.05) and ASR (0.47±0.03 v 1.10±0.06 mmol/L/d, p<0.01)compared to controls. Diabetics had a marked increase in GSH synthesisand concentrations with NAC(concentration 1.17±0.09 v 1.61±0.15, p<0.05;FSR 36±5 v 61±2, p<0.05; ASR 0.47±0.03 v 0.93±0.18, both supplements(concentrations 1.17±0.05 v 1.76±0.05, p<0.05; FSR 36±5 v 82±12, p<0.05;ASR 0.47±0.03 v 1.20±0.2, p<0.05). ROOH concentrations fell withn-acetylcysteine (13.2±1.7 v 5.78±0.78, p<0.05), and both supplements(13.2±1.7 v 4.65±0.31, p<0.01), and glycemia remained unchanged.

FIG. 6A demonstrates baseline data in diabetic vs. control subjects, andFIG. 6B shows GSH turnover in diabetics after precursor supplementation.FIG. 7 illustrates a synthesis pathway for glutathione.

Thus, in particular aspects of the invention, improving glutathioneconcentrations in hyperglycemic diabetic patients with glycine and NACsupplementation results in a profound decrease in damaging markers ofoxidative stress without a change in glycemia. The increased oxidativestress in diabetes is mediated by diminished GSH synthesis, due tolimited precursor availability. These findings are useful for theprevention and/or treatment of diabetic complications.

Example 4 Improving GSH Synthetic Rates and Concentrations in theElderly

Increased oxidative stress with aging has been linked to tissue damage,predisposing to many of the diseases common in the elderly. Theglutathione (GSH) redox system is a major component of antioxidantdefenses, and levels of protective GSH are lower in the elderly,indicative of impaired antioxidant defenses. Mechanisms responsible forglutathione depletion with aging remain unknown. It was considered thatdeficiency of precursor amino acids that comprise GSH, namely cysteineand glycine, predispose to decreased GSH synthesis, and oral dietarysupplementation with either precursor singly or in combination wouldaugment GSH synthesis, and decrease oxidative stress and plasma markersof ongoing oxidative damage.

Eight nondiabetic elderly humans were studied in the basal state, andafter 2-week oral supplementation with either n-acetylcysteine alone,glycine alone, or both supplements, with a 2-week washout periodin-between. All subjects received stable isotope infusions of²H₂-glycine and ²H₂-cysteine to measure the concentration and theabsolute synthetic rate (ASR) of red blood cell GSH. Plasma lipidperoxides were measured as a marker of ongoing damage due to oxidativestress. Eight younger adult subjects were also recruited as controls.

Elderly subjects (69.8±2.1y) had a significantly lower GSH concentration(mmol/L RBC) and ASR (mmol/L/d) compared to younger (40.5±3.4 y)controls (concentrations 1.02±0.2 v 1.73±0.11, p<0.01; ASR 0.57±0.18 v1.11±0.05, p<0.05. Elderly subjects had a marked increase in glutathionesynthesis and concentrations with n-acetylcysteine supplementation, butthe greatest response occurred with a combination of both supplements(Basal v post n-acetylcysteine: GSH concentration 1.02±0.24 v 1.37±0.30,p<0.05; GSH-ASR 0.57±0.18 v 0.92±0.23, p<0.05; Basal v postn-acetylcysteine and glycine: GSH-concentrations 1.02±0.24 v 1.53±0.24,p<0.01; GSH-ASR 0.57±0.18 v 1.08±0.11, p<0.05). Concomitantly there wasa significant fall in the concentrations of lipid peroxides (mmol/LROOH) with these supplements (5.10±1.03 v 3.22±1.29, p<0.01, and5.10±1.03 v 2.65±1.34, p<0.01 for n-acetylcysteine and both supplementsrespectively). Although the increase in GSH turnover with glycinesupplementation alone did not meet statistical significance (Basal vpost-glycine: GSH-concentrations 1.02±0.24 v 1.21±0.27; GSH-ASR0.57±0.18 v 0.71±0.05), this still resulted in a significant fall inROOH levels (5.10±1.03 v 3.57±1.13, p<0.05).

FIG. 8A shows basal GSH levels and precursor intake in young and elderlyhumans, and FIG. 8B shows GSH turnover in the elderly after precursorsupplementation.

Similar to aging humans, humans with uncontrolled diabetes also havevery low levels of glutathione, due to decreased synthesis -this leadsto unopposed oxidative stress and elevated markers of damage. Whenprecursors are provided in the diets, synthesis of glutathione increasessignificantly to that seen in non-diabetic humans, together with asignificant fall in oxidative stress and markers of oxidative damage(see FIG. 23).

These data demonstrate, for the first time, that increased oxidativedamage with aging in elderly humans is clearly linked to diminished GSHconcentrations, and the underlying mechanism is decreased GSH precursoravailability. Simple oral dietary supplementation with glycine andn-acetyl cysteine not only improves GSH synthetic rates andconcentrations, but also results in normalizing plasma markers of tissuedamage (lipid peroxides) to the levels of younger healthy controls.These results have a significant impact on the pathogenesis of oxidativedamage with aging: simple dietary interventions with GSH precursors canrestore GSH homeostasis and improve antioxidant status, and also haveimportant preventive effects on the chronic complications of aging.

Thus, elderly patients are deficient in glutathione, resulting inelevated oxidative stress. GSH levels are low due to decreasedsynthesis, due to low precursor availability. Markers of damage due tooxidative stress are elevated in a state of GSH deficiency. Precursorsupplementation improves GSH concentrations by elevating syntheticrates. The increase in GSH concentration reduces markers of damage dueto oxidative stress.

Example 5 The Role of Glutathione on Fat Oxidation, Weight Regulationand Insulin Resistance with Aging

The elevated risk of developing diabetes and cardiovascular disease withaging is associated with an increased incidence of insulin resistanceand obesity, but underlying mechanisms are poorly understood. Theinventor has found that compared to young healthy controls, aging isassociated with diminished synthesis and low intracellularconcentrations of glutathione (most abundant intracellular antioxidant)associated with increased oxidative stress and markers of oxidantdamage; correcting glutathione deficiency by dietary precursor aminoacid supplementation for 2 weeks results in a significant decrease inmarkers of oxidant damage. The mitochondrion is involved in both fattyacid oxidation and in the generation of superoxide free radicals in theelectron transport chain, and maintaining adequate mitochondrialglutathione levels is critical in preventing oxidant injury. Theinventor evaluated the effects of glutathione on fat oxidation, insulinresistance and body weight in healthy elderly humans and wild-type mice,in the basal glutathione depleted state, and after restoration ofglutathione levels by simple oral dietary supplementation withglutathione precursor amino acids.

This translational study has two arms: (a) Human: 8 young controls and 8non-diabetic elderly humans (60-75 years) were studied in the fastedstate with measurements of glutathione concentrations, markers ofoxidant damage, and respiratory gas exchange to compute fatty acidoxidation. (b) Animal: 8 young C57/B6 wild type mice (age 12-16 weeks),and two groups of 8 older (40-44 weeks) C57/B6 wild-type mice each werestudied. The young mice and one group of 8 older mice were fed a regularchow diet (5% fat calories) for 4 weeks, and the second group of 8 oldermice were fed an identical regular chow diet but with added supplementsof glutathione precursors cysteine and glycine for 4 weeks. All animalshad weekly weight measurements and were studied in the fasted stateafter the 4-week dietary intervention to measure respiratory gasexchange and energy expenditure by direct calorimetry; they also hadmeasurements of food consumption, glucose and insulin tolerance, andhepatic glutathione concentrations.

The results were as follows: (a) Humans: Elderly humans hadsignificantly lower red-cell glutathione concentrations compared toyoung controls (1.10±0.15 v 2.08±0.12 mmol GSH/L.RBC, p<0.05),associated with significantly higher levels of lipid peroxides andF2-isoprostanes. After a 16 h fast, elderly humans also had asignificantly lower fat oxidation (9.91±0.76 v 5.86±0.58 umol/kg/min,p<0.0001), and elevated plasma concentrations of fatty acids (0.48±0.01v 0.92±0.04 mEq/L, p<0.0001) and glucose (88±5 v 112±4 mg/dl, p<0.01).Glutathione precursor supplementation for 2 weeks in elderly humansresulted in a marked increase in the synthetic rate and red cellglutathione concentrations (1.10±0.15 v 2.07±0.31 mmol GSH/L.RBC,p=0.008), and falls in lipid peroxides (7.70±0.36 v 4.65±0.31mmol/L.ROOH, p<0.01) and F2-isoprostanes (136±12 v 85±14 pg/ml, p<0.05).There were collateral benefits in fat and glucose metabolism with astriking increase in fat oxidation (5.86±0.58 v 8.55±0.22 umol/kg/min,p<0.001), and), and significant decreases in fasting plasmaconcentrations of fatty acids (0.92±0.04 v 0.59±0.07 mEq/L, p<0.05) andglucose (112±4 v 93±3 mg/dl, p<0.05). (b) Animal: Compared to young WTcontrols, older mice had lower hepatic glutathione concentrations(8.4±0.5 v 4.1±0.2 umol GSH/g liver, p<0.05), lower fat oxidation(77.1±1.0 v 58.3±2.1, p<0.0000) and had poor glucose tolerance. With 4weeks of precursor supplementation, older mice had significant andstriking increases in hepatic glutathione concentrations (4.1±0.4 v5.7±0.3 umol GSH/g liver, p<0.05) and increased fat oxidation (58.3±2.1v 64.5±1.4, p<0.01). Older mice receiving glutathione precursorsupplementation lost 3.2% body weight (40.5±1.4 v 29.2±1.2 g, p<0.05)and improved insulin sensitivity, in contrast to the unsupplemented micewhich gained 13% weight (38.2±1.8 v 43.1±1.8 g, p<0.001).

FIG. 24 shows that improving GSH also results in increasing fatoxidation and decreasing Fatty acid concentrations in humans withuncontrolled diabetes.

These data indicate that there is a novel link connecting glutathionesynthesis and concentrations to fat metabolism, weight regulation andinsulin resistance. Glutathione deficiency in aging results in adiminished capacity to oxidize fat, leading to elevated fatty-acid andglucose levels, and insulin resistance. Stimulating glutathionesynthesis with simple dietary amino acid precursors significantlyimproves tissue glutathione concentrations. Improving glutathioneconcentrations results in increased fat oxidation, and leads to reducedfasting plasma fatty-acid and glucose concentrations, decreasedlipotoxic insulin resistance and decreased weight gain.

These observations indicate that glutathione deficiency occurs early inthe process of aging, and the innovative use of a safe and inexpensivedietary supplement to correct defective fat-oxidation and reducefatty-acid concentrations is a useful antioxidant therapy as a novelnutritional approach to combat at least insulin resistance, obesity anddiabetes in aging.

FIG. 9 illustrates exemplary study design for humans and animals. FIG.10 demonstrates exemplary measurements related to human studies forcharacterizing glutathione role on fat oxidation, weight regulation andinsulin resistance with aging. FIG. 11 provides the human studiesreferred to above in this example, and FIG. 12 provides the animalstudies referred to above in this example.

Example 6 Aging is Associated with GSH Deficiency

In certain aspects, aging is associated with Glutathione (GSH)deficiency. This leads to deficient capacity to oxidize fat, in turnleading to obesity, being overweight, and insulin resistance.

In a human study, the inventor has shown that feeding GSH precursorproteins in the diet improves GSH levels. The elderly humans had muchlower capacity to oxidize fat compared to young humans. After only 14days of consuming the GSH precursor proteins the elderly humans not onlynormalized GSH production and concentrations, but they also restoredtheir capacity to oxidize fat to that of young controls. This resultedin a 36% drop in their fatty acid levels.

Taking the concept to a mouse model, 3 groups of male mice were studied:young controls (14 weeks), and 2 groups of old controls (70 weeks). Theold mice were matched for age, weight and glucose tolerance. Both groupsof old mice received identical amounts of food, and the composition ofthe food was identical in terms of carbohydrate, fat and protein, exceptthat the treated group's protein was enriched with GSH precursors, andthe control group's protein did not have GSH precursors.

After 6 weeks of supplementation, the Rxd group decreased body weight by15%, which was entirely fat weight (no change in lean mass), increasedfatty acid oxidation, improved their glucose tolerance and insulinsensitivity together with a significant increase in liver and muscleGSH.

These data (provided in FIGS. 13-22) indicate that there GSHmanipulation results in significant metabolic benefits in terms ofinsulin resistance, dyslipidemia and body weight in mice due to increasefat oxidation. Early data from humans indicate that GSH manipulationresults in increased fat oxidation and improves dyslipidemia, and couldhave similar favorable benefits on insulin resistance and body weight.In specific embodiments, there is fat oxidation of ¹³C₁ palmitate,wherein the oxidation of a labeled fat as a direct measurement of fatoxidation, wherein old mice have a significantly lower oxidation of fatthan young mice.

Example 7 GSH and Neurodegenerative Disease in Aging

Improving GSH also results in decreasing F3 isoprostanes in elderlyhumans.

Since F3 isoprostanes are markers of oxidative stress mediated damage inthe brain, this has important implications for neurodegenerative diseasein aging (FIG. 25A). F2-isoprostanes are biomarkers of ongoing damagedue to oxidative stress.

Example 8 F2 Isoprostane Levels and GSH in Elderly Humans

Compared to young humans, elderly humans have increased F2 isoprostanesin the glutathione deficient state, indicating that there is increaseddamage due to oxidative stress (FIG. 25B). When glutathione synthesisand concentrations are corrected with dietary supplementation ofcysteine (as N-acetylcysteine) and glycine, the F2 isoprostane levels inelderly humans fall to that seen in younger humans, indicating thatthere is no further damage as a result of the protective actions ofglutathione.

Example 9 Role of Glutathione on Obesity, Insulin Resistance andDyslipidemia in Aging

In the present example and previous examples herein, the role ofglutathione on obesity, insulin resistance, and dyslipidemia in agingwas examined. Outcome measures include whole-body fatty acid oxidation,total body weight, fat and lean mass, glutathione concentrations inliver and muscle, energy expenditure, insulin resistance (from glucosetolerance tests), insulin simulated glucose disposal, total cholesteroland triglycride concentrations, and the exemplary molecular markersP-AMPK/AMPK, P-ACC/ACC, AND PGC1□.

FIG. 27 shows a study utilizing one group of young mice and two groupsof old mice. Of these old mice, one group is control (CON) and the othergroup receives the cysteine and glycine precursors. The graph shows thatold mice weigh significantly more than young mice, and that both groupsof old mice are identical in body weight. In particular cases, thesemice are employed in further studies described herein. Old mice do weighmore than young mice, and MRI data show that they have a higher fatcontent. The 2 groups of old mice were so randomized to be equal inweight so that the study could be done.

FIG. 28 shows basal whole-body fat oxidation in young mice, control oldmice, and experimental old mice that have not received treatment. Fatoxidation in old mice is significantly worse than young mice, and bothgroups of old mice are identical in terms of whole-body fat oxidation.

FIG. 29 shows fat oxidation by calorimetry. After six weeks, there areno significant changes in fat oxidation of young mice (see two bars onthe left). After six weeks, there are no significant changes in the fatoxidation of old mice, and this remains much lower than that seen in theyoung mice (see middle two bars). After six weeks of treatment withcysteine and glycine, there is a significant increase in the fatoxidation of treated old mice, and this reaches levels that are seen inthe young mice.

FIG. 30 illustrates the AMPK signaling cascade. FIG. 31 shows the AMPKcascade in the context of the invention. Phosphorylation of AMPK (i.e.an increased ratio of P-AMPK to AMPK) ultimately increases entry of LCFAinto the mitochondria. In FIG. 31, this occurs more in treated old micethan in untreated old mice. In FIG. 32, phosphorylation of ACC (i.e. anincreased ratio of P-ACC to ACC) ultimately decreases entry of LCFA intothe mitochondria, and the graph shows that treated old mice show thismore than untreated old mice. In FIG. 33, untreated old mice(unsupplemented) have lower PGC1a than young mice. After 6 weeks ofglycine+cysteine supplementation in the treated old mice, the PGC1aincreases, indicating that there are beneficial effects on mitochondrialbiogenesis.

In FIG. 34, treated old mice have a significantly improved insulinsensitivity (glucose-insulin product) compared to untreated old mice,and are similar to young mice following treatment. FIG. 35 shows¹³C₆-glucose oxidation, including the oxidation of a labeled glucose asa direct measurement of fat oxidation; old mice have a significantlylower oxidation of glucose than young mice. In FIG. 36, treated old micehave a significantly higher oxidation of glucose than untreated oldmice.

In FIG. 37, the Wt bars on the right show pre- and post-body weights intreated and untreated old mice and basal body weight in young mice. Thetreated old mice lose significant amount of weight compared to untreatedold mice. The lean body mass (LBM) bars show pre- and post-LBM intreated and untreated old mice and basal LBM in young mice. There is noloss of lean body mass in the treated old mice, indicating that the lossin body weight must be due to loss of fat mass.

FIG. 38 shows GSH concentrations in liver and muscle of young, controlold, and treated mice. Compared to young mice, the treated old mice(old-placebo group) have deficiency of glutathione concentrations in theliver and skeletal muscle of old mice. When supplemented with glycineand cysteine (as n-acetylcysteine) for 6 weeks, the treated old miceshow a significant improvement of glutathione concentrations within theliver and skeletal muscle. Thus, the studies described herein show areciprocal relation with GSH concentrations and fat oxidation withaging. Improving GSH concentrations with precursor supplementationresults in improving dyslipidemia, insulin resistance and obesity inmice.

FIG. 39 shows respiratory quotient (RQ) that is a measure thatindirectly reflects which substrate is being used as fuel for generatingenergy. The graph shows that young humans in the fasted state have an RQof 0.76, indicating that they are mainly burning fat to produce energy.Compared to young humans, the elderly humans in the fasted state have anRQ of 0.82, indicating that they are unable to burn fat to produceenergy as well as young humans. After two weeks of dietarysupplementation with glycine and cysteine (as n-acetylcysteine), elderlyhumans are able to significantly lower their RQ in the fasted state,indicating that they are now able to improve their ability to burn fatto produce energy.

FIG. 40 shows fat oxidation calculated from calorimetric data. In thebasal state, after a prolonged fast, elderly humans are not able tooxidize fat as well as young humans. After 2 weeks of dietarysupplementation with glycine and cysteine (as n-acetylcysteine) the sameelderly humans are now able to significantly increase fat oxidation inthe fasted state to that of young humans, indicating that the improvedlevels of glutathione is associated with an improved ability to oxidizefat to produce energy.

FIG. 41 shows fasting plasma fatty-acid concentrations in young andelderly. Compared to young humans, the elderly humans in the fastedstate have a much higher concentration of fatty acids in the plasma.After increasing glutathione concentrations (with two weeks ofsupplementation with n-acetylcysteine and glycine), plasma fatty acidconcentrations in elderly humans decreased by 35.7%.

FIG. 26 shows that improving glutathione concentrations also lowerstotal cholesterol and triglyceride levels in old mice.

FIG. 42 shows fasting plasma glucose in elderly humans after increasingglutathione concentrations (with 2 weeks of supplementation withn-acetylcysteine and glycine); fasting plasma glucose concentrations inelderly humans decreased by 10.5%.

Therefore, these studies indicate that improving GSH concentrations withprecursor supplementation is associated with improving fatty-acidoxidation and lowering fatty acid concentrations in humans. Improvingoxidation of fatty acids with improvements in glutathione concentrationsusing glycine and n-acetylcysteine supplementation is useful to targetobesity, dyslipidemia, insulin resistance and non-alcoholic fatty liverdisease in humans, for example.

Example 10 Glutathione in Treatment of Fibromyalgia and Statin-InducedMyopathy

FIG. 43 shows plasma concentrations of creatine kinase, which is abiomarker of muscle damage. CK levels after 6 weeks of study are shown.Treated old mice shows significant lowering of CK compared to untreatedold mice and young mice, indicating a beneficial effect of improvingglutathione.

Example 11 Impaired Glutathione Turnover and Increased Oxidative Stressand Oxidant Damage in Uncontrolled Diabetes and the Beneficial Effectsof Dietary Supplements with Glycine and N-Acetylcysteine

This example shows impaired glutathione turnover and increased oxidativestress and oxidant damage in uncontrolled diabetes and the beneficialeffects of dietary supplements with glycine and n-acetylcysteine. In anexemplary study, stable isotope infusions were used to study 12 diabeticand 12 healthy humans. The study measured GSH synthesis, erythrocyte GSHconcentrations, plasma oxidative stress, and oxidant damage.Concentrations of 1.33 mmol/kg/d for glycine and 0.83 mmol/kg/d for NACwere delivered for 14 days, for example.

FIG. 44 shows red blood cell glutathione concentrations. In the leftpanel, compared to nondiabetic humans, humans with uncontrolled diabeteshave deficiency of glutathione, a key antioxidant. In the right panel,providing dietary supplementation of cysteine (as n-acetylcysteine) andglycine in the diet significantly increases concentrations in humanswith uncontrolled diabetes. FIG. 45 shows GSH fractional synthesis rate.In the left panel, glutathione deficiency in humans with uncontrolleddiabetes occurs because of decreased synthesis. In the right panel,providing dietary supplementation of cysteine (as n-acetylcysteine) andglycine in the diet significantly increased glutathione synthesis andtherefore the concentrations in severely diabetic humans. Glutathionesynthesis rates were measured by the incorporation of labeled glycineinto glutathione, by using stable isotope tracers and gaschromatography/mass spectrometry.

FIG. 46 concerns oxidative stress and oxidant damage. Lipid peroxides(LPO) are biomarkers of oxidant damage, and DROMs are biomarkers ofoxidative stress. Compares to nondiabetic humans, humans withuncontrolled diabetes had increased LPO and DROMs in the glutathionedeficient state indicating that there is increased damage due tooxidative stress. When glutathione synthesis and concentrations werecorrected with dietary supplementation of cysteine (as n-acetylcysteine)and glycine, the levels of these markers fell significantly in diabetichumans, indicating that there was a significant decrease in diabeticdamage even though there was no decrease in the severely elevated bloodglucose levels.

Therefore, humans with poorly controlled diabetes have decreased GSHlevels because of diminished synthesis; synthesis is decreased due topoor availability of precursor amino acids. The low GSH statepredisposes one to increased oxidative stress, as measured by plasmamarkers of oxidative damage. Dietary supplementation with both NAC andglycine results in improved GSH synthesis and concentrations, and lowersoxidative stress and plasma markers of damage.

Example 12 Glutathione in Treatment of Impaired Fatty Acid Oxidation inHumans with Uncontrolled Diabetes

FIG. 47 shows improving GSH also results in increasing fat oxidation anddecreasing fatty acid concentrations in humans with uncontrolleddiabetes.

Example 13 Diabetic Mice: Energy Metabolism

Untreated db/db mice (unsupplemented) have lower PGC1a than nondiabeticmice. After 6 weeks of glycine+cysteine supplementation in the db/dbmice, PGC1a increases, indicating that there are beneficial effects onmitochondrial biogenesis/function and fatty acid oxidation (FIG. 48).

Thus, improving GSH concentrations with precursor supplementation isassociated with improving fatty-acid oxidation and lowering fatty acidconcentrations in humans. Improving oxidation of fatty acids withimprovements in glutathione concentrations using glycine andn-acetylcysteine supplementation is useful to target non-alcoholic fattyliver disease in diabetic humans.

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications herein are incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein their entirety.

PUBLICATIONS

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the claims. Moreover, the scope of the present application isnot intended to be limited to the particular embodiments of the process,machine, manufacture, composition of matter, means, methods and stepsdescribed in the specification. As one will readily appreciate from thedisclosure, processes, machines, manufacture, compositions of matter,means, methods, or steps, presently existing or later to be developedthat perform substantially the same function or achieve substantiallythe same result as the corresponding embodiments described herein may beutilized. Accordingly, the claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

The claimed invention is:
 1. A composition: (a) consisting essentiallyof glycine and N-acetylcysteine; or (b) consisting of glycine andn-acetylcysteine.
 2. A method of reducing deleterious effects ofoxidative stress in an individual, comprising the steps of providing aneffective amount of glycine and N-acetylcysteine to the individual. 3.The method of claim 2, wherein the individual is receiving treatment orhas received treatment for diabetes.
 4. A method of treating and/orpreventing diabetes in an individual, comprising the step of providingan effective amount of glycine and n-acetylcysteine to the individual.5. The method of claim 4, wherein the glycine and n-acetylcysteine areprovided to the individual in the same composition.
 6. The method ofclaim 4, wherein the glycine and n-acetylcysteine are provided orally tothe individual.
 7. A method of treating and/or preventing a medicalcondition in an individual, wherein the medical condition is associatedwith reduced glutathione levels, comprising the steps of: a) identifyingan individual in need of treatment of the medical condition; and b)providing an effective amount of glycine and N-acetylcysteine to theindividual.
 8. The method of claim 7, wherein the medical condition isdiabetes or complications of diabetes.
 9. A method of increasing GSHlevels in an individual in need thereof, comprising the step ofproviding an effective amount of glycine and n-acetylcysteine to theindividual, wherein the individual in need thereof is at least sixtyyears of age and/or has diabetes.
 10. A method of reducing body weight,lowering cholesterol level, and/or lowering triglyceride level in anindividual, comprising the step of providing an effective amount ofglycine and n-acetylcysteine to the individual.
 11. A method of treatingan individual for one or more medical conditions, comprising the step ofproviding an effective amount of glycine and n-acetylcysteine to theindividual, wherein the medical condition comprises: (a) dyslipidemiaand/or insulin resistance; (b) obesity; (c) fatty acid oxidation; (d)diabetic dyslipidemia; (e) elevated fatty acid levels; (f) diabeticmicrovascular complication comprising nephropathy, retinopathy, and/orneuropathy; (g) elevated cholesterol and/or triglyceride levels; (h)fatty liver disease; (i) neurodegenerative disease in aging; and/or (j)statin-induced myopathy.